The energy metabolism-regulating system in vivo consists of a food ingestion-regulating system and an energy consumption-regulating system. The energy consumption-regulating system participates in two categories of energy consumptions, that is, energy consumption for basal metabolism in order to sustain life and the other energy consumption. The main energy consumption under the latter category is nonshivering thermogenesis (nST), which is thermogenesis, and whose functional significance is maintenance of body temperature immediately after birth, during exposure to cold, at the end of hibernation, etc., and prevention of obesity and glycolipid metabolism disorder by consuming excess energy attributable to overeating, etc. Especially, in mammals and birds, which are homothermals, particularly in small animals, nST is important for maintaining body temperature. In the mechanism to induce nST, proton leak in mitochondria, that is, the participation of the electron transport system of the respiration chain in cells and ATP synthesis is important.
In a brown adipose tissue (BAT), a specific protein called an uncoupling protein 1 (UCP1) having a molecular weight of 32 kD and consisting of about 300 amino acids exists in the membranes of mitochondria. The protein has a function to uncouple the ATP synthesis from the electron transport system of the respiration chain of brown adipocytes (BA). UCP1 consists of a trebly repeating structure of a domain composed of approximately 100 amino acids and has 2 transmembrane regions in each domain, 6 sites in total. These transmembrane regions form channels in membranes of mitochondria.
UCP1 is a carrier that transports protons. Proton channels formed by UCP1 and others exhibit a function which allows protons to permeate freely in accordance with an electrochemical gradient to release heat. This is nST. That is, nST causes the uncoupling by proton permeation through proton channels, and the uncoupling reduces ATP synthesis, so that respiration in mitochondria is activated in order to keep the ratio of ATP to ADP constant. As a result, a large amount of fats and sugar are oxidized to generate heat.
The physiological significance of UCP1 is that it plays an important role in body temperature keeping immediately after birth, during exposure to cold, etc., and studies using transgenic mice have also elucidated that it participates in the prevention of obesity. The correlation of UCP1 with the development, progression and persistence of obesity has been suggested by the fact that there is a reduction in UCP1 expression in various obesity models. For example, it is confirmed that obesity develops without overeating in BAT-reduced transgenic mice (Lowell, et al., Nature, 366, 740-742 (1993)). Further, the reduction in body fat and the resistance against diet-induced obesity due to high fat-loaded meal were observed in mice which were forced to express a large amount of UCP1 by inserting UCP1 gene into a promoter of adipocyte-specific gene, aP2 (Kopecky et al., J Clin Invest, 96, 2914-2923 (1995)). Furthermore, the decrease in body temperature keeping function during the exposure to cold, obesity due to the increase of body fat, and insulin resistance were observed in mice whose UCP1 expression had been suppressed to ⅓(Lowell B. B., et al., Nature, 366, 740-742 (1993)). Further, UCP1 knockout mice were non-tolerant to cold (Enerback S. et al., Nature, 387, 90-94 (1997)). As shown above, it has become clear from animal experiments that UCP1 has important role in body temperature regulation and energy consumption as a thermogenetic molecule, and is closely related to obesity.
The amount of UCP1 expression is regulated mainly by the endonuclear gene transcription level, and UCP1 gene expression is increased by the elevation in cAMP concentrations (Saito et al., Saishin Igaku, 52, 1095-1096 (1997)).
Approximately 20 to 40% of intracellular energy consumption is considered to be produced by proton leak in mitochondrial inner membranes. Moreover, the majority of nST has been considered to be produced in skeletal muscles and white adipose tissues (WAT) in adult humans and other animals having little BAT. Based on the above-mentioned facts, it has been estimated that UCP exists in tissues other than BAT. cDNA cloning of UCP2 from other tissues than BAT was reported in succession by two groups in 1997 (Fleury, et al., Nature Genet, 15, 269-272 (1997): and Gimeno et al., Diabetes 46, 900-906 (1997)).
Human UCP2 shows 59% homology with human UCP1, and forms channels having 6 transmembrane regions as in UCP1, and it has purine nucleotide-binding sites. UCP2 differs from UCP1 in that it is widely expressed in the systemic tissue and is expressed in particularly high concentrations in the lung and pancreas, and expression is also detected in the heart, the liver, the brain, kidneys, testicles, WAT, BAT and skeletal muscles.
Regarding UCP2 function, the upregulation of UCP2 gene expression in adipose tissues around epididymids is observed in high fat diet-loaded mice. However, it was reported that UCP2 knockout mice were normal in body temperature keeping function under cold conditions (Arsenijevic D. et al., Nature Genet, 26, 387-388 (2000)). Further, an extensive upregulation of expression of UCP2 in the brown adipate tissue, which was considered compensation, was observed in the above-mentioned UCP1 knockout mice, and the mice were non-tolerant to cold (Enerback S. et al., Nature, 387, 90-94 (1997)). Further, it was shown that UCP2 suppressed insulin secretion via changing of intracellular ATP concentration in pancreas β cells (Zhang C.-Y. et al., Cell, 105, 745-755 (2001)). This is a disadvantageous property for diabetic therapy. As mentioned above, for UCP2, up to the present time, the relation to energy consumption/obesity has not been made clear although uncoupling function as proton channel has been confirmed.
The excessive energy in vivo is accumulated at first preferentially as visceral fat (especially, as mesenteric fat). Compared with fats at other sites (especially, subcutaneous fat), the visceral fat is apt to receive adipokinetic effect, and it is quickly decomposed and consumed. The visceral fat (obesity) is regarded as a multiple risk factor to cause life style-related diseases (adult diseases). The reason for this is that fatty acids secreted from white adipocytes (WA) in WAT are flown directly into the liver via the portal vein to accelerate insulin resistance and fat synthesis, and as a result, to induce sugar resistance abnormalities, high blood pressure and hyperlipemia, and these are finally complicated to cause arteriosclerosis. Accordingly, the inhibition of accumulation of the visceral fat and the reduction of accumulated visceral fat are expected to be effective for preventing the occurrence of life style-related diseases such as diabetics in adult humans and treating them.
A peroxisome proliferator activated receptor (PPAR) is considered as a member of a nuclear receptor (nuclear hormone receptor) super family from its structure and others. Up to now, three kinds of PPAR subtypes called PPAT α, PPAR δ (also called NUC-1, PPAR β or FAAR) and PPAR γ have been identified, and their genes (cDNA) have been cloned (Lemberger et al., Annu. Rev. Cell. Dev. Biol., 12, 335-363 (1996)).
It was reported that, fibrate agents having a ligand effect on PPARα, among the three kinds of PPARs, clinically show a strong lowering effect on serum triacylglycerol level (Forman et al., Proc. Natl. Acad. Sci. USA, 94, 4312-4317 (1997)).
PPAR γ is expressed especially in adipose tissues, and it was disclosed that the PPAR γ is a factor deeply implicated in regulating differentiation of adipocytes (Tontonoz et al., Genes and Development, 8, 1224-1234 (1994); and Tontonoz et al., Cell, 79, 1147-1156 (1994)). Various kinds of thiazolidinedione derivatives show a hypoglycemic effect in animal model of non-insulin-dependent diabetes mellitus (NIDDM) and are expected as new therapeutic agents for NIDDM having an insulin resistance breaking effect. A recent study demonstrated that the thiazolidinedione derivatives have also an effect as a ligand of PPAR y and activate specifically the PPAR γ (Lehman et al., J. Biol. Chem., 270, 12953-12956 (1995)).
However, physiological function of PPAR δ is not made clear yet (Willson et al., J. Med. Chem., 43 (4), 527-550 (2000)). WO97/28149 description disclosed that a PPAR δ ligand has a blood HDL increasing effect, and WO9904815 description disclosed that the administration of a PPAR δ-activating substance lowers a cholesterol level. But, there is no disclosure nor suggestion for the correlation between PPAR δ and a thermogenesis enhancing effect, and that between PPAR δ and an uncoupling protein.
Furthermore, it is known that an unsaturated fatty acid such as arachidonic acid, carbaprostacyclin (cPGI), and L-165041 (4-(3-(2-propyl-3-hydroxy-4-acetyl-phenoxy)propyloxy)-phenoxyacetic acid) increase the expression of UCP2 (The Journal of Biological Chemistry, Vol. 276, No. 14, Issue of April 6, pp. 10853-10860, 2001). However, there is no report on the correlation between UCP1 and PPAR δ.